Kinase suppressor of RAS (KSR) is a mitogen-activated protein kinase (MAPK) scaffold that is subject to allosteric regulation through dimerization with RAF. Studies in model organisms suggest that direct targeting of KSR could have important therapeutic implications for cancer therapy; however, testing this hypothesis through pharmacological approaches has been difficult owing to a lack of small molecule antagonists of KSR function. We have recently identified a promising set of lead compounds that bind directly to KSR to antagonize oncogenic RAS-MAPK signaling (Dhawan et al., Nature, 2016). This unique class of small molecules, which operate through stabilization of the KSR inactive state (KSRi), antagonize RAS-MAPK signaling by impeding RAF-KSR heterodimerization and conformational changes required for activation of the RAS-effector kinase MEK. Furthermore, we have found that KSRi has the potential to increase the efficacy of currently available MAPK inhibitors (including MEK inhibitors) by increasing therapeutic index, thereby expanding the potential utility of currently available MAPK inhibitors to treat patients with RAS-mutant cancers. Our preliminary studies have been based on in vitro studies, including reconstitution assays, cell line studies, and crystallographic analysis. In this proposal, we build upon our early leads so to create novel chemical probes that would be suitable for in vivo experiments. Compounds that emerge from this work will be valuable tools for the investigation of RAS-MAPK mechanisms, targets, and therapeutics. Furthermore, we will investigate direct targeting of KSR as a mechanism to inhibit deregulated RAS-MAPK pathway signaling within patient-derived and drug resistant cancer models. The RAS-MAPK pathway is activated in approximately 25% of human cancers, most often through point mutations in K-RAS. Deregulation of the RAS-MAPK pathway in patients is believed to be a major determinant of cancer initiation, progression, metastases, and often resistance to targeted therapies. Certain subtypes of cancer show a high percentage of RAS mutations; it is estimated that 95% of pancreatic cancers, 35% of colorectal, and 25% of lung cancers are dependent on mutant RAS-MAPK signaling. Currently, there are limited therapeutic options for these large patient populations. Small molecule targeting of KSR offers a novel approach to modulate RAS signaling pathways in disease, which if successful, could impact a high number of cancer patients.